The physical and biochemical effects of pre-rigor high pressure processing of beef.

High pressure processing (HPP) of pre-rigor longissimus thoracis (strip loin) from prime and bull animals substantially decreased the shear force and improved consumer eating attributes of the final meat product. The improved tenderness in both prime and bull meat was associated with a lower myofibrillar fragmentation index and reduced calpain 1 activity which indicated the mechanism of tenderisation was different from that which occurred in chill aged meat. Light microscopy showed disruption to the fibre packing within the muscle and electron microscopy confirmed significant disruption of the Z discs and M lines and disappearance of the A lines. Thus, HPP is associated with a reduction in the structural integrity and strength of the sarcomeres. These effects were consistent in strip loins sourced from prime and bull stock. HPP also led to the movement of glycogen phosphorylase from the sarcoplasmic fraction to the insoluble myofibrillar fraction in all animals and this was associated with a higher pH at 24 h.

High pressure processing improves the tenderness and quality of hot-boned beef.

Strip loins from different grades of cattle were subjected to two levels of high pressure processing (HPP) within 1h of slaughter at a commercial meat processing plant and chilled for 1day before freezing. The physical and eating quality characteristics of longissimus thoracis (strip loin) steaks from HPP were compared to meat that was chill aged for 1 or 28days. HPP produced meat after 1day with 60% lower shear force and higher sensory eating quality scores than 1day chill aged meat. Extended chill storage for 28days produced steaks of similar tenderness to HPP meat. HPP also increased the ultimate pH and decreased the cooking loss. Chilled storage of the gluteus medius from prime cattle for 28days significantly improved the shear force by 18%, whilst HPP improved both the shear force by 43% and the sensory eating quality scores. HPP can produce high eating quality eye of rump medallions within 1day of slaughter.

A macrocyclic calpain inhibitor slows the development of inherited cortical cataracts in a sheep model.

PURPOSE: We used sheep with an autosomal dominant gene for cortical cataract as an animal model to evaluate novel macrocyclic calpain inhibitors with potential for the medical treatment of human cataract. METHODS: The macrocyclic aldehyde, CAT811, identified previously as a calpain inhibitor that prevents calcium-induced opacification in cultured sheep lenses, was tested for its ability to protect cytoskeletal proteins from calpain proteolysis. CAT811 and its alcohol analogue, CAT505, were formulated separately into ointments, and each was applied twice daily to the right eye of sheep with early cataracts for five months. Progress of cataracts in the sheep was determined by ophthalmologic examination and comparison with a matched sample of sheep treated similarly with ointment that did not contain the active ingredient. RESULTS: The novel macrocyclic aldehyde, CAT811, was able to inhibit calpain proteolysis of lens cytoskeletal proteins at micromolar concentrations. When applied topically to the eyes of sheep, CAT811 was able to slow cataract development by 27% in the initial three months of treatment (P < 0.05). Its alcohol analogue, CAT505, was not able to slow cataractogenesis significantly. CONCLUSIONS: The inherited sheep cataract provides a reproducible model of cortical cataract over a time scale of several months. The data reported here, using this model, demonstrated the potential of the macrocyclic calpain inhibitor, CAT811, to act as a therapeutic for treatment of cortical cataract.

New tripeptide-based macrocyclic calpain inhibitors formed by N-alkylation of histidine.

Two new series of 15-membered macrocyclic peptidomimetics, in which the P1 and P3 residues of the peptide backbone are linked by a bridge containing a 1,4-disubstituted 1H-imidazole, are reported. The structure with an aldehyde at the C-terminus and the imidazole at P3, i.e., 4c, shows significant inhibitory activity against calpain 2, with an IC(50) value of 238 nM. The macrocyclic aldehyde with the imidazole at the alternative P1 position, i.e., 5c, is significantly less active. The relative activities are linked to the ability of the component macrocycles to mimic a ß-strand geometry that is known to favor active-site binding. This ability is defined by conformational searches and docking studies with calpain.

Molecular modeling: a search for a calpain inhibitor as a new treatment for cataractogenesis.

Studies of 17 analoges of 3 (SJA6017) in an in silico calpain model are reconciled to measured IC(50) values against ovine calpain. The studies validate the potential of the "model" and criteria established for inhibition as a tool to select structures for synthesis to test as calpain inhibitors. Using this screening methodology of virtual libraries led us to synthesize several inhibitors including macrocycle 33, which in vitro sheep eye lens culture experiments showed to substantially slow opacification.

Modulation of immune response to Toxoplasma gondii in sheep by immunization with a DNA vaccine encoding ROP1 antigen as a fusion protein with ovine CD154.

CD154 is a cell surface molecule expressed by activated T cells. CD40 and CD154 interaction is critically important in regulating humoral and cell-mediated immune responses. In this study we have investigated whether a DNA vaccine encoding rhoptry protein 1 (ROP1) of Toxoplasma gondii, and encoding ovine CD154 induces an enhanced ROP1-specific immune response in sheep. Two groups of twelve animals received two intramuscular injections, of a DNA plasmid encoding T. gondii ROP1 antigen (group 1) or an ROP1 antigen fused to ovine CD154 (group 2). There were two control groups of sheep. One was injected with an empty vector (group 3) and the other received no injections at all (group 4). The injection of the plasmid containing ROP1 (group 1) at weeks 0 and 4 induced a significant IgG2 response at week 2 which was amplified at week 4 after the booster injection and persisted to week 8 compared to the control animals in groups 3 and 4. For IgG1, significant differences from the control animals were only observed from week 5 onwards. The fusion of CD154 and ROP1 elicited significant IgG1 and IgG2 responses from week 1 which were amplified from weeks 5 to 8 compared to the control animals in groups 3 and 4. The IgG1 response was significantly higher in group 2 animals receiving pROP1-CD154 compared to group 1 receiving pROP1 only. There was no significant difference in IgG2 responses between groups 1 and 2. Significant differences in IFN-γ levels were only observed in treatment group 1 at week 2 and treatment group 2 at weeks 1 and 2 compared to the control animals. The results demonstrated that an intramuscular injection of pROP1-CD154 gene to sheep significantly enhanced their immune response and induced a mixed Th1/Th2 response while the intramuscular injection of pROP1 only induced a Th1-specific immune response.

Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii.

The dense granule proteins of Toxoplasma gondii are investigated as possible vaccine candidates against the parasite. The aim of this research was to evaluate the immune responses of sheep injected twice, intramuscularly, with DNA plasmids encoding T. gondii dense granule antigens GRA1, GRA4, GRA6 and GRA7 formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for GRA1, GRA4, GRA6 or GRA7 elicited an immune response after the first and the second injections as indicated by the moderate to high antibody responses. The injection of pGRA7 induced a significant level of anti-GRA7 IgG2 antibody and IFN-γ responses indicating a Th1-like immune response whereas injection with pGRA1, pGRA4 and pGRA6 stimulated a IgG1 type antibody response with a limited, if any, IFN-γ response. The results demonstrate that the intramuscular injection of sheep with a DNA liposome formulated plasmid coding for GRA proteins is an effective system that induces a significant immune response against T. gondii.

The immune responses of sheep after DNA immunization with, Toxoplasma gondii MAG1 antigen-with and without co-expression of ovine interleukin 6.

The aim of this study is to compare the immune responses of sheep stimulated by the intramuscular injection of a liposome formulated-DNA plasmid encoding the Toxoplasma gondii MAG1 antigen only or co-expressed with ovine IL-6. Forty-five, 2-year-old sheep were divided into four groups. Group 1 received an empty pVAXIg plasmid, group 2 no treatment, group 3 liposome formulated plasmid pVAXIgMAG1 and group 4, pVAXIgMAG1 plus pVAXovIL-6 plasmids. All the animals were inoculated at weeks 0 and 4. The injection of sheep with a plasmid encoding for MAG1 only or a MAG1 plasmid co-expressed with a plasmid encoding for ovine IL-6 produced humoral immune responses. The plasmids containing MAG1 elevated significantly serum IgG1 and IgG2 levels 2 weeks and onwards after the first injection of the plasmids. Co-expression of IL-6 with MAG1 had no effect on IG1 or IG2 levels illustrating that IL-6 in the formulation used had no modulating effect on any measured immune response.

Comparison of immune response in sheep immunized with DNA vaccine encoding Toxoplasma gondii GRA7 antigen in different adjuvant formulations.

Immunization with plasmid DNA, a relatively novel technique, is a promising vaccination technique. To improve the immune response by DNA vaccination various methods have been used, such as chemical adjuvants or immunomodulatory molecules formulated into microparticles or liposomes. The aim of this research is to evaluate the immune responses of sheep immunized with DNA plasmids encoding Toxoplasma gondii dense granule antigen GRA7 formulated into three different adjuvant formulations. Sixty sheep were injected intramuscularly with the DNA plasmids. Twelve received the liposome-formulated plasmid pVAXIgGRA7, 12 Emulsigen P formulated plasmid pVAXIgGRA7 and 12 Emulsigen D formulated plasmid pVAXIgGRA7. Twelve animals were used as a control and received the vector alone. All the animals were inoculated at week 0, and week 4. Immunization of the sheep with plasmids encoding GRA7, with the different adjuvant formulations, effectively primed the immune response. After the first inoculation, moderate to high antibody responses were observed with the three different adjuvant formulations. A significantly elevated specific IgG2 response was observed in the sheep immunized with liposomes and Emulsigen D as adjuvants. In the group immunized with Emulsigen P as an adjuvant, lower IgG1 and IgG2 antibody levels were developed compared to the other treatment groups. In all the immunized groups, DNA immunization stimulated a IFN-gamma response. No antibody or IFN-gamma responses were detected in the control group immunized with an empty plasmid or not immunized. These results indicate that intramuscular immunization of sheep with a DNA vaccine with the adjuvants liposomes and Emulsigen D induce a significant immune response against T. gondii.

Molecular modeling, synthesis, and biological evaluation of macrocyclic calpain inhibitors.

The design and elaboration of a series of macrocyclic templates that exhibit a propensity to adopt a beta-strand-like peptide-backbone conformation led to potent and selective inhibitors of calpain 2. Macrocycle 1 retarded calcium-induced opacification in an ovine-lens culture assay and is a lead compound for the development of a drug for cataract treatment. Cbz=carbobenzyloxy.

The involvement of calpains in opacification induced by Ca2+-overload in ovine lens culture.

OBJECTIVE: To investigate biochemical changes accompanying Ca(2+)-induced lens opacification and the possible role of calpain activation in opacification within an ovine lens culture system. METHODS: Sheep lenses were cultured in minimal media. Lens opacification was induced by exposure to the Ca(2+) ionophore, ionomycin, and graded by digital image analysis. Cell viability was estimated by the release of lactate dehydrogenase into the culture medium. Opaque lenses were fixed and stained for a microscopic view of the lens structural changes. Ionic changes in the lens were measured by atomic absorption spectroscopy. Calpain activation was determined by zymography on casein gels and proteolysis was investigated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE) and Western blotting. The calpain inhibitor, SJA6017, was used to investigate the involvement of calpains in lens opacification. RESULTS: Treatment of cultured ovine lenses with ionomycin increased total lens Ca(2+) concentration and caused the cortical region of the lens to become opaque. Addition of the Ca(2+) chelator, EGTA, inhibited the ionomycin-induced changes. Progress of opacification correlated with the death of lens cells and lens swelling in differentiating fiber cells. Autolysis of calpain 2, following ionomycin treatment, suggested activation of this protease. 2DE revealed that the ionomycin did not result in substantial proteolysis of the crystallins. However, Western blotting revealed significant breakdown of the cytoskeletal proteins, spectrin and vimentin. The pattern of the breakdown products was consistent with calpain proteolytic activity. SJA6017 retarded the cortical opacity induced by Ca(2+)-overload in the ovine lens. CONCLUSION: The ovine lens with Ca(2+)-induced opacification by ionomycin is associated with calpain activation and the subsequent proteolysis of cytoskeletal proteins. These events could be initial factors contributing to cell death and the loss of lens transparency which occurs in this ovine model of cataractogenesis. The ovine model supports the hypothesis that cytoskeletal proteins and Ca(2+) homeostasis play an important role in maintaining lens transparency.

Synthesis, biological evaluation and molecular modelling of N-heterocyclic dipeptide aldehydes as selective calpain inhibitors.

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.

Evaluation of a novel calpain inhibitor as a treatment for cataract.

PURPOSE: The aim of this study is to evaluate the therapeutic potential of a newly synthesized calpain inhibitor, CAT0059, using a naturally occurring in vivo sheep cataract model. METHODS: The selectivity of CAT0059 was investigated by an in vitro protease assay. The efficacy of CAT0059 in preventing proteolysis of lens cytoskeletal proteins by calpain 2 was investigated using a lens-based cell-free method. The cytotoxicity and stability of CAT0059 in physiological conditions were examined using cultured sheep lenses. Protein binding of CAT0059 by ocular proteins was assessed and quantified by a modified high-performance liquid chromatography assay. CAT0059 was formulated in an eye drop solution and as an eye ointment. These were applied in vivo daily to one eye of the cataract lambs, over a 67- and 97-day trial period, respectively. The progression of cataracts in the treated and untreated eyes was assessed by an independent veterinary ophthalmologist using a slit-lamp microscope. RESULTS: In vitro assays revealed that CAT0059 was selective for cysteine proteases and also protected lens cytoskeletal proteins from degradation. CAT0059 was stable in physiological conditions and non-toxic to the lens. Only 15% of CAT0059 is bound to proteins in the aqueous humour but >90% bound to lens homogenate. The 67-day CAT0059 eye drop treatment was not effective in slowing the rate of cataract development. However, application of CAT0059 in an eye ointment initially slowed cataract development compared with the untreated eye. This effect was temporary. CONCLUSIONS: In vitro assays confirmed CAT0059 to be a potent calpain inhibitor. The two in vivo trials addressed the ability of CAT0059 to reach the lens and established its limitations as a therapeutic molecule for cataract treatment.

Calpain may contribute to hereditary cataract formation in sheep.

PURPOSE: To determine the involvement of calpain in ovine cataractogenesis by measuring calcium, calpain activity, proteolysis, and the effect of calpain inhibition. METHODS: Sheep with genetic cataracts were examined for cataract severity. Calcium in normal and cataract lenses was measured. The presence of calpain was detected by casein zymography and immunoblotting. Calpain activity was assayed using BODIPY-casein as a substrate. Degradation of calpain substrates spectrin and vimentin was assessed by immunoblotting. The calpain inhibitor SJA6017 was applied to the left eye of cataract lambs, leaving the right eye as an untreated control. Both eyes were monitored by slit-lamp microscopy for cataract progression. RESULTS: Cortical cataracts were first observed in lambs at 1 to 2 months of age. Lens calcium concentration increased in the early stages of cataract formation and was >10-fold higher in mature cataract than normal lenses. Three calpain isoforms were detected in young lamb lenses. Calpain activity decreased as cataracts progressed. Both spectrin and vimentin were degraded with cataract maturity, which could indicate calpain proteolysis. Cataract lambs treated with SJA6017 eyedrops over a period of 4 months showed significantly smaller cataracts in the left treated eye over the right untreated eye. CONCLUSIONS: The presence of calpains and calcium elevation during cataract formation suggests that proteolysis may play a role in opacification in ovine lens. This hypothesis is supported by the delay in opacification with SJA6017 treatment. The results also suggested that the ovine hereditary cataract is a useful nonrodent model to test the role of calpains in cataractogenesis.

Up- and down-regulation of longissimus tenderness parallels changes in the myofibril-bound calpain 3 protein.

The objective of this study was to utilize Ca(2+) and Zn(2+) treatments of meat to critically explore the possible role of calpain 3 in meat tenderisation. Calpains 1 and 2 were also examined for comparative purpose. Control animals plus animals infused with CaCl(2), ZnCl(2) or H(2)O were used (six lambs per treatment) to determine the temporal changes in muscle calpain 3 protein in the Longissimus thoracis et lumborum (LTL) during post-mortem storage. Concurrently, the temporal changes of; (1) shear force, (2) sarcomere length, (3) proteolysis of titin and nebulin and (4) calpains 1 and 2 proteins were also determined. Infusing LTL with Ca(2+) or Zn(2+) caused significant up- and down-regulation of LTL tenderisation, respectively, compared to water infusion and the control animals. Furthermore, the rate of breakdown of calpain 3, the rate of proteolysis of titin and nebulin and the rate of meat tenderisation during post-mortem storage of LTL in the various treatments were highly correlated. These studies suggest that calpain 3, like calpain 1, may be involved in the tenderisation of meat through limited proteolysis of specific muscle structural proteins such as titin and nebulin.

Postmortem changes in myofibrillar-bound calpain 3 revealed by immunofluorescence microscopy.

An immunofluorescence microscopy method for following changes in myofibrillar-bound calpain 3 was developed. Afterward, proteolytic changes in calpain 3(p94), calpain 1, titin, and nebulin were examined in myofibrils prepared from ovine longissimusthoracis et lumborum (LTL) stored for 0, 1, 2, and 3 days postmortem. Western blot analysis revealed that the levels of intact calpain 3 (expressed as percentage of the level immediately postmortem) were 80%, 10% and not detectable in myofibrils prepared at 1, 2, and 3 days, respectively. Western blots for calpain 1 also indicated conversion of the intact protein (80 kDa) to a 76 kDa fragment during the same time period. Thus calpains 1 and 3 appear to be activated during postmortem storage. Immunofluorescence microscopy using an IS1 region specific antibody revealed that calpain 3 staining was most intense at the sarcomere Z- and M-lines. The fluorescence intensity declined significantly during storage, paralleling changes in the proteolytic breakdown of titin and nebulin associated with these structures.

Does the newly discovered calpain 10 play a role in meat tenderization during post-mortem storage?

The objective was to study calpain 10 during meat tenderization. Western assays were developed and changes in calpain 10, tenderization level and desmin were determined in Longissimui (LTL) during post-mortem storage. A comparison between some characteristics of calpains 1, 2 and 10 indicated differences in the pI and sub-cellular distribution. Tenderness improved gradually during post-mortem storage and was accompanied by a decline in intact desmin level. Western analysis indicated that calpain 10 is present in sheep LTL probably as two proteins (MW 73.6 and 70.7 kDa). Post-mortem storage caused a decline in the level of the 73.6 and 70.7 kDa proteins in the sarcoplasmic fraction and a concomitant increase in these proteins in the myofibrillar fraction. Changes in the myofibrillar calpain 10 proteins were strongly correlated with the rate of tenderization. To conclude, calpain 10 proteins are present in sheep LTL and the sub-cellular distribution is dynamic during post-mortem storage.

The relationship between meat tenderization, myofibril fragmentation and autolysis of calpain 3 during post-mortem aging.

The objective was to study the potential role of calpain 3 in postmortem myofibril breakdown and meat tenderization. We determined the temporal changes in calpain 3 protein in the ovine m. longissimus thoracis et lumborum (LTL, n=4) during post-mortem storage. Concurrently, we also determined the kinetics of tenderization level, changes in MFI, degradation of nebulin and desmin, and autolysis of calpain 1. The autolysis of calpains 1 and 3 were strongly correlated with the kinetics of tenderization and changes in MFI. The best correlation was between the appearance of the autolyzed calpains 1 and 3 and nebulin degradation. Taken together, the results indicated that calpains 1 and/ or 3 might be playing a key role in post-mortem tenderization of LTL via the proteolysis of specific muscle structural proteins such as nebulin. This is the first report that relates calpain 3 to myofibrillar protein degradation in post-mortem skeletal muscle.

Particulate metmyoglobin reducing activity and its relationship with meat color.

There is controversy about the effect of storage time on metmyoglobin (MetMb) reducing activity in the sarcoplasmic fraction of meat and the role of this reducing activity in maintaining the color of red meat. The presence of metmyoglobin reducing activity in the myofibrillar fraction of muscle extracts was investigated as a possible reason for this controversy. NADH-dependent MetMb reducing activity was found in the particulate fraction of meat following sedimentation of a meat homogenate at 35 000g. There was 5.8 times more MetMb reducing activity in the particulate fraction compared with that of the sarcoplasmic (supernatant) fraction. The presence of metmyoglobin reducing activity in the myofibrils (MMRA), defined as the activity in the sediment after centrifugation of a meat homogenate at 2 000g, was confirmed. The myofibrillar fraction contained 63% of the total MetMb reducing activity available in the meat. The relationship between muscle MetMb reducing activities and meat color parameters was investigated in a beef patties system. The particulate MetMb reducing activity (PMRA) in beef patties was found to be a good indicator of the total metmyoglobin reducing activity in meat and was positively correlated with the color parameters of the patties, suggesting that PMRA may be associated with red meat color of meat patties.